Therapeutic blood clotting factor compositions and their use

ABSTRACT

Compositions including, in units/ml, factor VIII correctional activity, about from 2-35; prothrombin, about from 1-10; thrombin, less than about 0.003; factor VII, about from 37-190; factor VIIa, about from 8-80; total factor IX, about from 15-112; factor IX precursor, 0 to about 30; factor X, about from 1-30; and factor Xa, about from 1-10 are used in therapeutically effective amounts to treat blood clotting factor inhibitors.

This is a division of application Ser. No. 262,286, filed May 11, 1981,now U.S. Pat. No. 4,357,321 which in turn is a division of Ser. No.116,186, filed Jan. 28, 1980 and now U.S. Pat. No. 4,287,180.

BACKGROUND OF THE INVENTION

This invention relates to the treatment of blood coagulationpathologies. The invention generally deals with novel methods forpreparing new compositions useful in the management of hemorrhagicepisodes in patients with inhibitors of blood clotting factors. Inparticular, the invention is concerned with the therapy ofantihemophilic factor or plasma thromboplastin component inhibitors.*

Blood coagulation is an exceedingly complex process. The interaction ofvarious blood components which eventually gives rise to a fibrin clothas been compared to a cascade of steps, each of which is dependent uponand regulated by preceding and following steps. Generally, the bloodcomponents which take part in the coagulation cascade are eitherproenzymes or enzyme modulators. The proenzymes are enzymaticallyinactive proteins which are converted to proteolytic enzymes by theaction of an "activator", generally another proteolytic enzyme producedat an earlier stage in the coagulation cascade. Coagulation factorswhich have undergone such a conversion are hereafter defined asactivated factors, and designated by the lower case postscript "a" whilethe proenzymes are referred to as precursor clotting factors.

The enzyme modulators are principally cofactors such as calcium ions ornonenzyme proteins and most are essential if the enzymes are to exhibitany catalytic activity at all. Such modulators are to be distinguishedfrom enzyme substrates. Substrates are compounds which are covalentlymodified by an enzyme while modulators or cofactors merely bind to theenzyme without undergoing a change in structure.

Blood coagulation is best visualized as a cascade of reactions betweenformed and soluble blood components in which most segments of thecascade are demarked by a proenzyme. An initial event such as thecontact activation of Hageman factor (factor XII) will start one branchof the cascade. Basically, the product of this initial event thenactivates the next proenzyme in the cascade and so on in a sequentialprocess until a fibrin clot is formed, the total rate of the cascadebeing dependent upon cofactors and modulators.

While much is now known about the various blood coagulation factors andthe manner in which they interact and are susceptible to environmentalinfluences, there remain many areas where questions remain. One sucharea is the action, etiology and therapeutic treatment of blood clottingfactor inhibitors.

Inhibitors of blood clotting factors pose substantial difficulties inconventional hemorrhage therapy. Uncontrolled bleeding due tocoagulation deficiencies is usually halted by supplying the deficientcomponent from pooled plasma sources. However, since inhibitors arefrequently elucidated by the patient in response to the presence of thedeficient clotting factor, this conventional approach is frequentlycounterproductive since increasing the dosage of clotting factor merelyresults in greater output of inhibitor. This problem with clottingfactor inhibitors is not an isolated one. For example, up to anestimated 21% of the Hemophilia A population develops factor VIIIinhibitor, i.e., antihemophilic factor (AHF) inhibitor. To fullyunderstand the background of this invention it is necessary to discussthe mechanisms of blood clotting and the influence of inhibitorsthereon.

The etiology of such clotting factor inhibitors is not well defined, butis thought to be along two principal routes. First, as explored above,frequent infusions of therapeutic clotting factor concentrates such asAHF frequently produce an immune response in patients as evidenced byincreased inhibitor titer. Multiple challenges of the patient's immunesystem with AHF are believed to stimulate ever-increasing AHF antibodylevels. Since such antibodies may then complex with the AHF and blockits activity, the increases in antibody titer dictate greater doses toachieve a satisfactory clinical response.

In contrast, the second route of appearance for the inhibitors is notbelieved to be a function of the administration of therapeutic bloodprotein fractions. Rather, the inhibitor seemingly arises spontaneouslyin the manner of an idiopathic or autoimmune disease, frequentlyfollowing on the heels of drug reactions or collagen disorders.

The medical community has dealt with clotting factor inhibitors by (a)administering either extremely low or extremely high doses of theclotting factor which is being inhibited, with or withoutimmunosuppression, (b) using clotting factor of non-human origin or (c)administering activated prothrombin complex concentrates (PCC), i.e.,PCC in which at least a small proportion of the clotting factors havebeen converted to active enzymes. The first two techniques have not beenwidely used. The infusion of sufficiently large amounts of clottingfactor to overwhelm the inhibitor existing in the patient's systembecomes less and less effective with each treatment episode becauseinhibitor titers rise in response to each administration of clottingfactor. On the other hand using non-human clotting factor creates a riskof severe immune reactions in treated patients.

The use of activated PCC for the treatment of patients afflicted withclotting factor inhibitors has received widespread acceptance, followinga presentation by Fekete et al. at the XIV International Congress ofHematology in 1972. For example, see Kurczynski et al., "New EnglandJournal of Medicine" 291(4):164 (1974) wherein an activated PCCcontaining 15 units of factor II/ml (the term "factor" will befrequently abbreviated herein as "F"), 200 units of F-VII/ml, 42 unitsof F-IX/ml, 58 units of F-X/ml, 3-10 units of F-IXa/ml, 3-8 units ofF-Xa/ml and 0.001-0.003 units of thrombin/ml was used therapeutically totreat F-VIII inhibitor afflicted patients. The clinical success of suchconcentrates has been ascribed to the presence of the various activatedcotting factors VIIa, IXa of Xa, or thrombin, although the identity ofthe operative activated factor or factors is subject to controversy.Recently, success has also been attributed to the presence of acomponent possessing "factor eight inhibitor bypassing activity"(FEIBA). This component is not believed to be one of the activatedfactors II, VII, IX or X, but otherwise the nature of its activity isnot well defined.

Activated PCC, when used to treat factor-VIII inhibitor, has theadvantage that the complex can be tailored to be sufficiently free offactor VIII antigen that an immune response in humans are not observed.

The activated factors which are present in most of the prothrombincomplex concentrates previously used to treat clotting factor inhibitorsare artifacts of the plasma fractionation procedure in which prothrombincomplex is enriched from Cohn fraction I supernatant; the activatedfactors were not induced by any special steps and as a result were oftenconsidered to be in too low or too variable a concentration to besatisfactory.

While processes for the activation of PCC have been generally alluded toin the art, the only detailed disclosure known to applicants of aprotocol for manufacturing such products appears in U.S. Pat. No.4,160,025, to Eibl et al. These patentees urge that before their methodwas developed, activated prothrombin complex concentrates "could not betested as regards their effective principle and could not bestandardized . . . therefore the results [were] not safe and [could]hardly be repeated." The patentees go on to state that their method "hasas its object to safeguard in a repeatable and deliberate manner ageneration of the desired factor-VIII-inhibitor-bypassing activity" (twoparagraphs bridging columns 1 and 2).

The Eibl et al. method comprises activating a starting material selectedfrom plasma, cryoprecipitate-poor plasma or Cohn fraction I supernatantby use of a contact activator, followed by adsorption of the FEIBAcomponent and factors II, VII, IX and X onto a basic ion exchanger.Contact activators are well known substances such as silica or kaolinwhich initiate the intrinsic coagulation mechanism by activation ofHageman factor.

While Eibl et al. are highly concerned with standardizing their finalproduct they give scant attention to the activation procedure. The pH,temperature, starting materials and activators are generally describedbut no mention is made of the activation period other than the one orthree hours disclosed in Examples 1 and 2.

It is extremely difficult to avoid excessive activation of prothrombincomplex concentrates because the activation reactions, being an enzymecascade, tend to accelerate rapidly at variable and largelyunpredictable rates which are controlled by substances in the activatedsample and by the kinetics of the enzymes in question. The mostpotentially harmful result of excessive activation is the appearance ofthrombin, or activated factor II in the product. For example, Eibl etal. report thrombin levels of 0.05 and 0.07 NIH units/ml. Thrombin isnot considered desirable because it is capable of acting directly onblood components to yield a fibrin clot while other activated clottingfactors exert their effect earlier in the coagulation cascade and henceare more likely to be subject to modulation by blood components in vivo.The elevated thrombin levels reported by Eibl et al. are believed byapplicant to be a function of the failure of Eibl et al. to adequatelycontrol the activation procedure. Eibl et al. do not screen the startingmaterial for activation, thus failing to taken into account thepre-existing activation state of each lot of plasma or plasma fractionused, and do not determine the in-process response of the lot toactivation. However, Eibl et al. do suggest in Example 1 that variationsin the clotting factor and FEIBA levels in various lots of final productmay be compensated for by mixing bulk batches until the desired ratio ofclotting factors to FEIBA is achieved. This is unsatisfactory because ofcosts, yield losses and contamination risk inherent in such a procedure.Further, thrombin is frequently undesirably elevated, even in productswhich were apparently manufactured by following this procedure.

According to White et al., "Blood" 49 (2): 159-170 (1977), American RedCross PCC is nonthrombogenic in part because of the presence of heparinand the deliberate fortification with antithrombin III. These twoadditives are said to result in the irreversible inactivation ofproteases in PCC. White et al. did not report treating inhibitorpatients with such PCC; it is problematic that such a concentrate wouldbe useful for this purpose where any activated factors which mightinadvertently be present, as well as mechanisms for their generation,are suppressed by heparin and antithrombin III.

It is accordingly an object of this invention to standardize activatedPCC preparations without mixing or handling the finished product.

It is an additional object to control the manufacture of activated PCCto reduce the production of thrombin during the process and toneutralize any thrombin which is produced.

It is another object to treat patients having clotting factor inhibitorsor deficiencies with an activated PCC containing selected activities offactors II, VII, total IX, X, VIIa, IX precursor, and Xa.

These and other objects of the invention will be apparent to thoseskilled in the art from consideration of the specification taken as awhole.

The principal object of this invention is accomplished by determining inadvance of the completion of activation the conditions needed to achievean activated PCC of substantially predetermined composition. This is incontrast to the passive approach to the unsolved problems ofstandardization and thrombogenicity which characterizes the publishedprior art, where conditions such as the time and temperature ofactivation are arbitrarily set and any difficulty with the resultingproduct is remedied, if possible, by selecting lots which when combinedwill yield the desired products. Accordingly, in a method wherein aprothrombin ciomplex-containing blood protein fraction is activatedunder conditions which produce enzymatically active blood clottingfactors, the improvement comprises

(a) selecting at least one of said conditions which is to be varied tocontrol the degree of activation;

(b) prior to the completion of activation, determining the magnitude ofthe condition needed to activate the fraction to a predetermined degreeof activation;

(c) setting the condition to said magnitude; and

(d) conducting the activation of the fraction in accordance with saidcondition.

Generally only one condition of the activation is permitted to vary, andthis is usually the period of time that activation is allowed toproceed.

The magnitude of the selected condition is determined in one of twoways, or a combination of both. In the least preferred of the twomethods, the condition is determined by removing aliquots of thefraction after activation has been commenced, terminating the activationof each aliquot, determining the degree of activation of each aliquotand calculating the magnitude of the condition necessary to achieve apredetermined degree of activation of the fraction.

Alternatively, the condition magnitude may be determined by removingaliquots of the fraction prior to activation, varying the conditionamong the aliquots, activating the aliquots in accordance with thecondition set for each aliquot, terminating the activation, determiningthe degree of activation of each aliquot and calculating the magnitudeof the condition necessary to achieve a predetermined degree ofactivation of the fraction. This embodiment has the advantage than onecannot overrun the predetermined activation level, as could be doneduring the assay of aliquots withdrawn from a bulk lot which issimultaneously undergoing activation.

Control of the activation process is also facilitated by selecting asstarting materials only fractions which exhibit a low degree ofspontaneous activation.

The degree of activation is generally monitored by following thenonactivated partial thromboplastin (NAPT) or factor VIII correctionaltimes although thrombin determinations are also useful. These assays arefully described below. The levels of individual clotting factors, mayalso be determined as a measure of activation. Methods for determiningthese factors are also described therein.

An additional object of this invention is accomplished by activating anintermediate PCC produced during the method of U.S. Pat. No. 3,560,475.Selection of a particular point in the patented method to activate thePCC greatly facilitates the control of the activation procedure becauseof the presence in the PCC of an activation retardant.

A further object is achieved by adding heparin to the activated productafter the activation retardant has been neutralized or removed,generally immediately before the product is filled into containers andlyophilized.

A further object is accomplished by including the stabilizers heparinand, optionally, antithrombin III in the final activated PCC. Thesesubstances inactivate thrombin and are believed to provide a margin ofsafety against thrombosis in susceptible patients, e.g., those withliver dysfunction.

This invention also includes an improved activated PCC composition whichcomprises an aqueous solution having clotting factor activities, inunits/ml, of F-II, 1-10; thrombin, less than about 0.003; F-VII, aboutfrom 37 to 190; F-VIIa, about from 8 to 80; total F-IX, about from 15 to112; F-IX precursor, 0 to about 30; F-X, about from 1 to 30; and F-Xa,about from 1 to 10. More particularly, the improved activated PCC willcontain certain delineated levels of total F-IX and F-IX precursor,F-VII and F-VIIa, F-X and F-Xa, F-VIII correctional activity and NAPTtime, and will be sufficiently free of factor VIII antigen to notproduce an immune response in patients to whom the activated PCC isadministered.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Suitable starting materials for use herein should at least containclotting factors II, VII, IX, X, XI and XII. The starting compositionswill generally be solutions of Cohn plasma fractions I+II+III, I andIII, II and III, III, III-0, IV-1 and IV-4; IV-1 is preferred. Thecompositions should be dissolved in buffer or saline to a concentrationof about 10% weight/volume at about 20° C. and then screened forclotting factor activity as described below to determine the degree ofpre-existing spontaneous activation. The coagulation factors are thenpartially purified by adsorption onto a suitable known prothrombincomplex adsorbent, e.g., tribasic calcium phosphate, as described inU.S. Pat. No. 3,560,475, or a diethylamino ethyl group substitutedresin, followed by elution from the adsorbent in a volume of elutingsolvent equal to about 4% of the volume of the dissolved Cohn fraction.None of these volumes or temperatures are critical.

The starting materials are then preferably assayed to detect any whichmight have inadvertently been spontaneously activated to a high degree.Whether or not a starting composition is suitable is determined byassaying the NAPT time and, preferably, the factor VIII correctionaltime of the fraction. The former is a conventional assay, disclosed forexample by Pepper et al., "British Journal of Haematology" 36:573 (1977)or Kingdon et al., Abstract #86 of the meeting of the American Societyof Hematology, Atlanta (1974). It is preferred that the startingmaterial be diluted within the range of 1:10 to 1:1000 in Tris bufferedsaline, optimally 1:100, before determination. If the sample is used atfull strength the clotting time is often excessively rapid. Since theNAPT time has as its readout signal the formation of a plasma clot, thetest results are usually reported in seconds when a 1:100 dilution isused. All references to the NAPT time herein are for assays on 1:100dilutions of the sample or standard in a solution of 0.06 M Tris insaline at pH 8.3 (hereinafter Tris buffered saline).

The factor VIII correctional assay comprises the following steps, allconducted at 37° C. Aliquots of the composition to be tested are dilutedin a barbital buffer to give dilutions of 1:20, 1:40, and 1:80. Thisbarbital buffer is a modification of the diluting fluid (described inProctor, et al., Am.J.Clin.Path. 36(3):214 (1961) which is made bymixing one part of diluting fluid with one part of water. 1:20, 1:40 and1:80 dilutions of reference activated PCC having one unit of F-VIIIcorrectional activity/ml are made into the diluting fluid as with theunknowns. For the purposes herein, 1 unit of factor VIII correctionalactivity is defined as that quantity of a 1:20 dilution into dilutingfluid of activated PCC which, upon addition to factor VIII deficient orinhibitor plasma having less than 5% of the factor VIII activity ofnormal human plasma, will correct the clotting time of that plasma to 35seconds under the conditions of the above assay. Reagent blanks areprepared in the same fashion as the standards.

In the conduct of the assay, 0.1 ml of a mixture of soluble ellagic acid(available commercially from Dade under the trademark Actin) is added toa set of prewarmed fibrometer reaction cups. 0.1 ml of factor VIIIdeficient plasma having less than 5% of the factor VIII activity ofnormal pooled human plasma is then added to each cup. 0.1 ml each ofaliquot, blank or standard dilution is added immediately to a cupcontaining the mixture of ellagic acid and factor VIII deficient plasma.After three minutes 0.1 ml of 0.02 M CaCl₂ is added to each cup toinitiate clotting. The clotting time is recorded and corrected for thereagent blank clotting, if necessary.

The factor VIII correctional activity in units is calculated byaveraging the replicates and plotting the reference concentrations, asestablished by the dilutions, against the respective clotting times. Theconcentration of each diluted sample can be located from the plot,corrected for its dilution and the average concentrations reported inunits/ml. It is preferred to simply report the factor VIII correctionalassay results in seconds where screening the starting materials ordetermining activation conditions. However, the potency of the finalproduct is generally reported in units/ml. When screening the startingfractions, dilutions are made in a non-modified diluting fluid.

Starting materials having a NAPT time of greater than about 200 secondsand a factor VIII correctional time of more than about 89 seconds areacceptable for use in the process of this invention. For example, in agroup of 30 Cohn fraction IV-1 pastes, each of which was prepared from adifferent plasma pool, the NAPT time ranged from 144 to 294 seconds andthe factor VIII correctional time from 82.7 to 98 seconds. It ispreferred to screen the starting materials for thrombin as well, and, insuch cases, starting material should not be used if it containssufficient thrombin to form a clot within 2 hours in the assay describedbelow, i.e., less than about 0.001 units/ml of thrombin. Also, thestarting materials should contain about from 0.4 to 1.0 units/ml ofprothrombin, about from 0.5 to 3.0 units of F-VII/ml, 0.5-1.5 units ofF-IX/ml and 0.5-3.0 units of F-X/ml.

The effectiveness of feedback assays to establish the extent ofactivation can be improved by slowing the rate of activationsufficiently to allow a generous period in which to conduct the assays.This reduces the chance of exceeding the predetermined activation statewhile performing the assays. One convenient technique for slowing theactivation rate is to activate the coagulation factors in the presenceof a plasma component which will be referred to hereinafter as anactivation retardant. The activation retardant slows the rate ofactivation and is defined as a substance which is removed or neutralizedduring the process of adsorbing a 10% solution of Cohn fraction IV-1paste onto 0.5% by weight of tribasic calcium phosphate at pH 7.2,eluting from the calcium phosphate with 0.1M sodium citrate andprecipitating from the calcium phosphate eluate at a PEG concentrationof 5%. The identity of retardant is unknown, but has been hypothesizedto be antithrombin III or unidentified diluent proteins which slow therate of activation. The amount of antithrombin III remaining in thecalcium phosphate eluate is about 1 International Unit/ml, while theproduct after PEG precipitation contains about 0.1 Unit/ml.

Once a suitable starting material preferably containing the activationretardant has been selected, a procedure is instituted to convert atleast inactive proenzyme to the corresponding active blood clottingfactor, hereinafter the activation procedure. This is conventionallydone by contact activating plasma or plasma fractions. This isaccomplished by mixing a contact activator such as kaolin, silica orsilicates with the starting material and continuing to mix until thedesired activation state is achieved. Contact activators are well knownand the selection of any one is not critical. However, it is preferableto use an insoluble activator so as to facilitate its removal when thedesired degree of activation is accomplished. Silica is preferred. Thecontact activator is used in a concentration of about from 0.05 to 5%weight by volume preferably about 0.06%. The average temperature ofactivation may range from 0° C. to about 30° C., and preferably is about15° C. The pH may range about from 5.5 to 8.5, but is preferably about7.2. The protein concentration ranges about from 0.3 to 0.9 gm%.

The extent of activation is usually controlled by holding all of theseconditions constant but one, and varying that one to yield thepredetermined degree of activation. It is preferred to control theactivation period by holding pH, temperature and other conditionsconstant while varying the reaction time. This is convenient because thereaction is readily terminated by centrifuging or filtering the reactionmixture, preferably by filtration through a cartridge filter having 1.2micron pores. However, it is within the scope of this invention to holdthe reaction time constant but vary another condition, e.g., if a bulklot needed little activation the reaction could be conducted at a lowertemperature than with a lot needing more vigorous treatment. Activationcontrol by temperature offers an additional advantage in that at lowertemperatures, i.e., 0° C. to about 10° C., the production of factor VIIais favored in comparison to thrombin and factors IXa and Xa. Finally,more than one condition may be varied, but this generally is notpreferred.

The activation period will depend upon the extent of activation desiredcompared to that which has already occured spontaneously in the startingplasma as determined by screening the starting material. The preferreddegree of activation, expressed in NAPT time is about from 70 to 100seconds, preferably 75-95 seconds. The preferred degree of activationmay also be expressed as a factor VIII correctional time of about from70-90 seconds and preferably about 70-80 seconds. The factor VIIIcorrectional time is not preferred for monitoring activation statebecause the change in clotting time during activation is not as large asthat usually encountered in the NAPT time. It is within the scope ofthis invention to use the NAPT time to the exclusion of the factor VIIIcorrectional time assay as well as the thrombin generation time or FEIBAtests disclosed by Pepper et al., op cit. The clotting times reportedherein, unless otherwise stated, are for samples immediately afteractivation. Further concentration of the activated PCC will reduce theNAPT and factor VIII correctional times, e.g. generally doubling theconcentration of clotting factors will approximately halve the NAPTtime. Further, a different degree of activation as represented bydifferent predetermined NAPT and factor VIII correctional times may beselected depending upon the clinical uses to which the final product isto be put. Also, the degree of activation may be determined by otherassays, e.g., one or more of the activated clotting factor testsdescribed below. The actual elapsed time per se is generally notmaterial, but has been found to range about from 5 to 45 minutes,routinely 15 minutes.

The preferred method for ascertaining the magnitude of time needed foractivation for each individual bulk lot of starting material comprisesfirst determining the activation period using aliquots withdrawn fromthe bulk lot before activation, and then monitoring the lot duringactivation as well. The first portion of this preferred method comprisesremoving a plurality of samples from the bulk lot before activation,activating each sample and then stopping the activation at differenttimes. Each aliquot is subsequently assayed for its NAPT and factor VIIIcorrectional times, a plot of the results is made and the period whichmust elapse for the attainment of the desired NAPT, and optionallyfactor VIII correctional times, is determined. Also, it is preferredthat thrombin be determined as well. The bulk lot is then activated withthis period in mind under the same conditions as were used with thealiquot samples. Aliquots of the lot during activation are also taken toconfirm its progress.

In more detail, the first portion of the preferred embodiment comprisesremoving duplicate 110 ml aliquots of sample, mixing about 60 mg ofsilica with each aliquot at a temperature of 15° C.-20° C., allowing theactivation to proceed for 5 minutes and multiples thereof up to 60minutes, filtering the aliquots to separate the silica, recording thetime of activation for each aliquot, and determining the average NAPTand factor VIII correctional times for each aliquot. The activationperiod required to attain NAPT and factor VIII correctional times,respectively, of about from 70-100 seconds and about from 70-90 secondsis then determined by interpolation. This entire procedure willordinarily require less than 1.5 hours. During this time the bulk lotfrom which the samples were withdrawn cam be simply held at 15°-20° C.without any significant changes in the levels of activated factors.

The second portion of the preferred method for determining the properactivation time has the advantage that the effect of the activationprocedure on the bulk lot itself is monitored, yielding a more directresult than following the activation of aliquots that are intended to berepresentative. This portion of the method comprises commencingactivation of the screened starting material, withdrawing 10 ml samplesfrom the reaction mixture at 5 minute intervals and assaying each forfactor VIII correctional and NAPT times and, optionally, thrombin. Whenthe predetermined factor VIII correctional or NAPT time is projected tobe reached within a next 5 minute interval, the activation procedure isstopped by filtrative removal of the silica. It is desirable toextrapolate the assay results because the assays measure the state ofactivation at the time the sample is taken, and not at the time theresults are read. The projection is conventionally based upon a plot ofthe factor VIII correctional and NAPT times observed earlier during theactivation procedure. Any risk that the activation will overshoot thetarget parameters during the assays is small if the assays areexpeditiously conducted.

Following the completion of controlled activation the clotting factorsmay be further purified to any desired degree, although it is notnecessary to do so. It is preferred that the activated PCC be purifiedand concentrated by PEG precipitation as disclosed in U.S. Pat. No.3,560,475 and discussed above. Other protein isolation techniques may beused, e.g., adsorption on ion exchange resins, gel chromatography orprecipitation by such well known agents as alkanols or Pluronicpolymers.

The purified product is dissolved in a volume of aqueous solution equalto about 2% of the fraction IV-1 paste solution. This aqueous solutionpreferably contains 1 volume of 0.1M sodium citrate, 4 volumes of 0.9%NaCl and from 1 to 2 units of heparin/ml. The pH is adjusted to aphysiologically tolerable level, for example 7.0, clarified and sterilefiltered by conventional techniques, dispensed into vials andlyophilized. The product is generally reconstituted into sterile waterto yield the same concentration as before lyophilization. Thereconstituted product will contain from about 0.5 to 1.5 units ofheparin/ml, preferably greater than about 1.1 units/ml.

Heparin should be added at the final dissolution of the activated PCCbefore lyophilization, although it may be added at any point afteractivation. It is preferable that the heparin be added just prior tolyophilization. Antithrombin III may also be added at this point,although since the product will ordinarily contain about 0.1International unit of antithrombin/ml it may be unnecessary to add moreantithrombin III. If necessary, sufficient antithrombin III and heparinare added to reduce any extraneous thrombin activity in thereconstituted activated PCC to a level lower than about 0.003 units ofthrombin/ml.

Clotting Factor Assays

The analytical methods for determining factors XI, XII, II, VII, IX, X,Xa, IX precursor, VIIa and thrombin are generally conventional. All ofthe assays have certain features in common, unless otherwise specified.First, each assay will include making duplicate serial dilutions of testsample and a standard having an assigned potency of 1 unit/ml. Theconcentration in units/ml of the test sample may then be calculated byaveraging the duplicates, plotting the results obtained with thestandards against their respective percent concentrations as establishedby their previous serial dilution, reading the percent concentrations inthe diluted test samples from the plot, correcting the test sampleconcentrations for the serial dilutions which were made; averaging thetest sample percent concentrations and dividing the average by 100 toarrive at the units/ml of the assayed factor.

Second, unless otherwise indicated all assays are conducted at 37° C.and all reagents are prewarmed to that temperature.

Third, assays for total clotting factors use either lyophilized normalhuman plasma or frozen normal human plasma as standards. The lyophilizednormal human plasma is standardized against three separate freshly drawnpools of normal human plasma. Each pool is prepared by collecting venousblood from 10 fasting, normal donors who are not taking oralcontraceptives, anti-inflammatory drugs or arthritis medication. Thedonors must also have a prothrombin time of 11-15 seconds, an APTT of30-45 seconds and a fibrinogen level of 200-400 mg/dl. The blood iscollected into 3.8% sodium citrate at a ratio of 9 volumes of blood to 1of anticoagulant, mixed, centrifuged at 1000 RCF for 15 minutes, afterwhich equal volumes of each plasma supernatant are pooled. The plasma isassayed within one hour. The average potency of the three pools for eachtotal factor assayed below is arbitrarily set at 1 unit/ml.

The frozen normal human plasma is prepared in identical fashion to anyone of the three freshly drawn pools described above, except that thepool is distributed in 1 ml volumes into plastic vials and frozen at-70° C. The frozen pools are used within 60 days. Each frozen pool isconsidered to contain 1 unit of each total factor/ml. Hereinafter,plasma which contains standard unitage as established by either of thetwo foregoing techniques will be referred to as reference or standardplasma.

Fourth, the factor deficient plasma used in some assays are plasmaobtained from donors that are congenitally deficient in the particularfactor, i.e., who have a factor potency of less than about 5% of thatpresent in normal pooled plasma.

Fifth, the F-IX assays detect total and precursor F-IX. The assay fortotal F-IX measures the sum of activated and unactivated F-IX activity,while the F-IX precursor assay substantially excludes the activatedmaterial. Therefore, F-IXa may be estimated by subtracting the precursoractivity from the total F-IX. It should be noted that the remaininganalytical methods, i.e., for factors II, VII, X, XI and XII, allmeasures the sum of active and proenzyme factor. However, in theinterests of brevity the designation "total" will not be applied tothese assays. On the other hand, and in contrast to the F-IX methods,the thrombin, VIIa and Xa methods disclosed below directly assay theactive factors.

Thrombin is determined by the following technique. A bovine thrombinstandard, which has been standardized against the NIH Thrombin Standard,lot B-3, is diluted in normal saline to 0.001, 0.002, 0.003, 0.005 and0.010 u/ml. 2.0 ml of this diluted standard is added to 0.5 ml offibrinogen substrate. The mixture is incubated at 28° C. The reactiontubes are checked every 2 minutes. First fibrin strand appearance istaken as the end point. The test sample is assayed identically, but withno dilution. Thus, 2.0 ml of the reconstituted test sample is added to0.5 ml of the fibrinogen substrate and end point formation is observedat 2 minute intervals. The clotting times of the test sample arecompared with the clotting times of the thrombin standard. Thecalculations are conducted as generally described above.

Factor Xa is determined by a modification of the method of Yin et al.,"J. Lab. Clin. Med." 81:298(1973). All reagents, including the referencestandard, are commercially available from the Sigma Chemical Company.Test samples are serially diluted in duplicate into the buffer employedby Yin et al. at dilutions of 1:8, 1:16 and 1:32, or higher (expressedin parts of sample to parts of buffer) until the clotting time of thatdilution is longer than the clotting time of the factor Xa standard at aconcentration of 0.01 units/ml.

Standard factor Xa is initially diluted 1:4 into the same buffer,followed by serial dilutions in duplicate to 1:64. A 1:4 dilution ofstandard F-Xa is taken as 1 unit F-Xa/ml. Standard F-Xa is defined asthat which will produce an average clotting time of 14 seconds at 1:2dilution in the assay described herein. 0.1 ml of each final dilution ispipetted into a fibrometer cup, followed by 0.1 ml of 0.025M CaCl₂ and0.2 ml of a bovine plasma-rabbit cephalin solution to initiate clotting.The clotting time for each tube is determined and the F-Xa activitycalculated as described above.

F-Xa may also be determined by a chromogenic assay as an alternative tothe clotting method described in the preceding paragraph. Unlessotherwise stated by designation of the assay results as "chromogenic" itwill be assumed that the F-Xa was determined by the clotting method. Thechromogenic assay is essentially disclosed by Kosow in "ThrombosisResearch" 1:565-573 (1976). It employs a synthetic substrate which isspecifically hydrolyzed by F-Xa to yield a chromogen detectable by itsadsorption of light at 405 nm. The substrate, S-2222, is commerciallyavailable from Ortho Diagnostics, Inc. Standard F-Xa is available fromthe Sigma Chemical Co., but is diluted 1:4 into 0.05M Tris buffer at pH8.3 containing 1.33% NaCl by weight before use. A 1:4 dilution of astandard containing 0.5 unit F-Xa/ml should exhibit an average opticaldensity at 405 nm of 0.260 in the assay. In the practice of the assay,samples and diluted standard are serially diluted into the Tris buffer.0.4 ml of each dilution is pipetted into a glass test tube, followed by0.075 ml of a solution containing 0.5M CaCl₂ and 0.1M NaCl and, after 1minute, 0.5 ml of an S-2222 solution in 0.05M Tris buffer at pH 8.3containing 0.9% NaCl by weight. 0.1 ml of 50% acetic acid is added after3 minutes to stop the reaction and the absorbance is read against abuffer blank at 405 nm. The calculations are conducted as generallydescribed above.

Factor X is determined by a modification of the Bachmann et al. methoddescribed in "Thromb. et Diath." 2:24(1958) except that factor Xdeficient plasma is used in place of Seitz filtered ox plasma, afibrometer is used for end point detection and the diluting fluid isveronal buffer containing sodium chloride and sodium citrate asdescribed by Proctor et al., "Am. J. Clin. Path." 36(3):214(1961).Russell's viper venom and cephalin were obtained from Burroughs Wellcome& Co. and the Hyland Division of Travenol Laboratories, Inc.,respectively. The calculations are made as generally described above.

Prothrombin (factor II) is assayed by the following technique. 0.1 ml offactor II deficient plasma prepared by the method of Pechet inTocantins, Ed., Blood Coagulation, Hemorrhage and Thrombosis, volume 1,pp 144-148 (1964) is distributed into each of eight test tubes. A 100%reference plasma is prepared by diluting reference plasma 1:10 into1.72% imidazole weight/volume buffer at pH 7.3. This reference plasma isthen further diluted 1:5, 1:10, 1:20 and 1:40 into the same buffer.Duplicate 0.1 ml aliquots of each dilution are pipetted into the testtubes containing factor II deficient substrate. Immediately afterpipetting the reference plasma into each duplicate set of test tubes,0.2 ml of rabbit brain thromboplastin lyophilized with CaCl₂ is added toeach test tube by means of a plastic-tipped pipette. After mixing for 15seconds each tube is tilted back and forth once per second over a lightsource and the time that elapses before final gel formation is recorded.The foregoing procedure is repeated with the test sample, except that a1:100 dilution into imidazole buffer is made before the 1:5, 1:10, 1:20and 1:40 dilutions. The data is handled in the same fashion as discussedabove.

F-IX is determined by the following procedure, essentially that ofProctor et al., op cit. A minimum 1:20 predilution of the activated PCCtest sample is prepared in normal saline. Reference plasma is notprediluted. Then duplicate 1:5, 1:10, 1:20 and 1:40 dilutions inbarbital buffered saline of test sample and reference plasma arepipetted into test tubes already containing 0.1 ml of partialthromboplastin-kaolin described in the Proctor et al. procedure and 0.1ml of F-IX congenitally deficient plasma having less than 5% of normalF-IX activity. After 3 minutes, 0.1 ml of 0.03M CaCl₂ is mixed with thecontents of each test tube, incubated for 30 seconds and then each testtube is tilted at less than once per second in front of a light sourceuntil final gel formation. The time from CaCl₂ addition to gel formationis recorded and the data treated as generally described above.

F-IX precursor is assayed exactly as set forth above for total F-IXexcept that the initial minimum 1:20 dilutions of test sample are madeup in the F-IX deficient substrate rather than normal saline.

Factor VII is determined according to Esnouf et al. in Bang et al., Ed.,"Thrombosis and Bleeding Disorders, Theory and Methods," pp 197-198,(1971) except that the clotting point was determined with a Clotek®device and the diluting fluid was that described by Proctor et al. opcit.

Factor VIIa is assayed by first adsorbing the sample with abenzamidine-Sepharose affinity matrix. The benzamidine-Sepharose matrixis a well known affinity gel disclosed, for example, by Schmer, "Z.Physiol. Chem." 353: 810-814 (1972). The non-adsorbed fraction isremoved from the matrix by washing with 0.1M NaHCO₃, ph 7.8. Then, thesame buffer containing 0.5M NaCl and 0.3M benzamidine HCl is used toremove the fraction containing VIIa. Assay of the latter fraction forVIIa is acomplished with the same assay and reference which are used forF-VII.

The assay for factor XI is described by Rappaport et al. in "J. Lab.Clin. Med." 57:771(1961), except that the CaCl₂ solution is 0.03M, acephalin-kaolin mixture commercially available from the Hyland Divisionof Travenol Laboratories, Inc. was employed, and the clotting point wasdetermined with a Clotek® device.

Factor XII is determined in essentially the same way as factor XI.However, here a factor XII deficient plasma is used and the assay isonly conducted in contact with plastic ware.

The novel activated products of this invention are characterized by theamounts or activities of individual clotting factors, overallprocoagulant activity as reflected in the NAPT and F-VIII correctionaltimes, substantial freedom from thrombin activity and substances whichinduce an immune response to F-VIII in treated patients, greater than 1unit of heparin/ml and the presence of about 0.1 to 3 units antithrombinIII/ml in the final product. Combinations of any or all of the foregoingfeatures also characterize the products of this invention.

The typical, preferred and most preferred ranges of clotting factoractivities in the activated PCC of this invention fall within the limitsset forth in Table 1 below. In addition to the components set forth inTable 1 the products may optionally also contain about from 3 to 65units of F-XI and 1 to 30 units of F-XII/ml.

                  TABLE 1                                                         ______________________________________                                        ACTIVATED PCC CLOTTING FACTOR LEVELS                                                     Range in units/ml                                                  Factor       Typical  Preferred  Most Preferred                               ______________________________________                                        II           1-10     3.6-8.9    3.6-5.9                                      VII          37-190    37-122    39-88                                        VIIa         8-80     25-78      25-60                                        IX           15-112   20-81      50-80                                        IX Precursor 0-30      5-20       5-12                                        X            1-30      1-25       1-13                                        Xa           1-20      1-10       4-10                                        Xa (Chromogenic)                                                                           1-10     1-8        1-5                                          Thrombin     0.003    0.002      0.001                                        ______________________________________                                    

Obviously the ranges for each of these factors expressed in units/mlwill depend upon the reconstitution volume of the activated PCC, whichmay be varied depending upon the intended use for the product. Thus theabove ranges in units/ml may also be considered the relativeproportionate units on a dry weight basis, the actual concentration inunits per milliliter depending upon the reconstitution volume of theactivated PCC. The ranges given above are for activated PCC which isdiluted or reconstituted for direct administration to a patient.

A preferred composition of this invention comprises, in units/ml, aboutfrom 20 to 112 units of F-IX and from 0 to about 30 units of F-IXprecursor.

Another preferred compositions of this invention comprises, in units/ml,about from 37 to 190 units of F-VII and about from 25 to 80 units ofF-VIIa.

A further preferred composition of this invention comprises, inunits/ml, about from 1 to 13 units of F-X and about from 4 to 10 unitsof F-Xa.

This invention also includes a product comprising factors VIIa, IXa andXa and having a F-VIII correctional activity of about from 1 to 35units/ml and a NAPT time of about from 27 to 70 seconds/ml. Thepreferred composition exhibits a F-VIII correctional activity of aboutfrom 7 to 30 units/ml.

The activated PCC of this invention may be administered to patients inthe same fashion as PCC has heretofore been administered.Conventionally, the contents of vials containing lyophilized, activatedPCC are reconstituted in sterile water and infused at a therapeuticallyeffective dosage, generally ranging about from 8 to 160 F-VIIIcorrectional units/kg and preferably about from 10 to 80 F-VIIIcorrectional units/kg. Optimal results are obtained with dosages ofgreater than about 25 F-VIII correctional units/kg, preferably 50units/kg. If required, the dosage may be repeated at 6 to 8 hourintervals. Total dosages of activated PCC on rare occasions have rangedup to about 2000 F-VIII correctional units/kg; satisfactory therapy isusually seen at total dosages of about from 8 to 300 F-VIII correctionalunits/kg, preferably about from 10 to 100 F-VIII correctional units/kg.Since total dosage refers to the quantity of F-VIII correctionalactivity administered during a bleeding episode rather than the amountadministered during any one infusion, it can be seen that one infusionis frequently effective in achieving clinically satisfactory results.

One embodiment of therapeutic treatment is the administration of atherapeutically effective dose of an aqueous composition comprisingabout from 20 to 112 units of F-IX/ml and from 0 to about 30 units ofF-IX precursor/ml to a patient exhibiting a clotting factor inhibitor.

Another embodiment of the therapeutic method of this inventioncontemplates the administration of a therapeutically effective dose ofan aqueous composition comprising about from 37 to 110 units of F-VII/mland about from 8 to 80 units of F-VIIa/ml to a patient exhibiting aclotting factor inhibitor.

An additional embodiment of this invention is the administration of atherapeutically effective dose of an aqueous composition comprisingabout from 1 to 50 units of F-X/ml and about from 4 to 10 units ofF-Xa/ml to a patient exhibiting a clotting factor inhibitor.

This invention will be more fully understood by reference to thefollowing examples.

EXAMPLE I Manufacture of Activated Prothombin Complex

This example discloses a typical manufacturing run for the controlledpreparation of an activated PCC.

80 kgs of Cohn fraction IV-1 paste are suspended in 720 liters of salineand the pH adjusted to 7.2 with 1N sodium hydroxide. A resultant heavysediment is allowed to settle, after which a clear supernatant isobtained by centrifugation. The NAPT and factor VIII correctional timeswere determined to be 240 and 98 seconds, respectively. As these timesfall within the criteria of greater than 200 and 89 seconds,respectively, the lot was selected for activation. 3.6 kgs of calciumphosphate are added to the clarified supernatant. After 15 minutesmixing the suspension is centrifuged to recover the calciumphosphate-absorbed coagulation factors. The factors are separated fromthe calcium phosphate by vigorous mixing for 5 minutes with a volume of0.1M sodium citrate equal to 4% of the dissolved IV-1 paste volume. Thesuspension is centrifuged and the supernatant recovered.

The coagulation factors in the supernatant are then activated by adding20.9 gm of silica to the supernatant and continuously mixing. 10 mlsamples are withdrawn at 5 minute intervals and the degree of activationdetermined by the NAPT and factor VIII correctional times, and thrombinassays described above.

The silica induced activation is terminated by filtration of thereaction mixture through a 1.2 micron cartridge when the NAPT and factorVIII correctional times reached 90 and within 70-90 seconds,respectively. The thrombin activity at this point was below 0.003units/ml.

The activated PCC is next further purified by the PEG precipitationsteps disclosed in U.S. Pat. No. 3,560,475. The filtrate from the silicaremoval step is brought to 5% weight/volume PEG by the addition of 1.4kg PEG having an average molecular weight of 4000 (PEG-4000). Thesuspension is centrifuged after mixing for approximately 15 minutes, itspH adjusted to 5.2 and the supernatant brought to 20% PEG-4000 by thefurther addition of 4.1 kgs of PET. The suspension is centrifuged aftermixing for approximately 15 minutes, and the precipitate collected. Theprecipitate is dissolved in 0.02M sodium citrate containing 0.72% NaCland 1.5 units heparin/ml, the pH adjusted to 7.0, clarified, sterilefiltered, filled into 30 ml vials and lyophilized. The factor VIIIcorrectional and NAPT activities and the levels of clotting factors inthis preparation reconstituted in water are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                                          Activity (in units/ml                                       Factor or Activity                                                                              unless otherwise noted)                                     ______________________________________                                        II                7.8                                                         VII               47.6                                                        VIIa              58.8                                                        IX                39.2                                                        IX Precursor      11.5                                                        X                 9.3                                                         Xa                1.6                                                         Thrombin          0.002                                                       Factor VIII correctional                                                                        21.2                                                        NAPT time (1:100 dilution)                                                                      45.6 (seconds)                                              ______________________________________                                    

EXAMPLE II

The process of Example I was substantially repeated on 10 more lots ofPCC. The results are shown in Table 3 below.

                                      TABLE 3                                     __________________________________________________________________________    RESULTS OF TESTS PERFORMED ON FINAL CONTAINERS OF 10 LOTS OF ACTIVATED        PCC                                                                           (All potency values expressed as units/ml except as otherwise noted)                            F-VIII    F-IX              APT                             Lot Number                                                                           F-II                                                                             F-VII                                                                             F-VIIa                                                                            Correction                                                                          F-IX                                                                              Precursor                                                                           F-X                                                                              F-Xa                                                                             Thrombin                                                                            (Seconds)                       __________________________________________________________________________    0650D010                                                                             7.1                                                                              63.4                                                                              77.8                                                                              25.8 (11.6.sup.a)                                                                   66.7                                                                              8.6   11.1                                                                             7.2                                                                              0.002 27.3                            0650D012                                                                             3.6                                                                              39.0                                                                              25.4                                                                               7.7.sup.a                                                                          23.2                                                                              6.1   10.4                                                                             3.5                                                                              0.001 41.1                            0650D013                                                                             7.1                                                                              47.0                                                                              58.8                                                                               9.0.sup.a                                                                          40.3                                                                              6.3   12.5                                                                             5.2                                                                              0.001 39.7                            0650D014                                                                             7.2                                                                              65.9                                                                              35.2                                                                               9.9.sup.a                                                                          55.2                                                                              10.3  10.2                                                                             4.1                                                                              0.001 39.7                            0650D018                                                                             5.6                                                                              68.3                                                                              32.5                                                                               8.8.sup.a                                                                          54.8                                                                              10.3  14.7                                                                             4.8                                                                              0.001 37.3                            0650D021                                                                             5.3                                                                              65.5                                                                              40.1                                                                              14.5  34.7                                                                              8.3   11.0                                                                             2.2                                                                              0.001 54.4                            0650D024                                                                             8.9                                                                              63.5                                                                              36.0                                                                              27.6  112.3                                                                             36.3  24.2                                                                             7.6                                                                              0.001 38.9                            0650E021                                                                             3.8                                                                              71.9                                                                              25.8                                                                              16.7  70.1                                                                              5.0   10.4                                                                             6.8                                                                              0.001 34.1                            0650D028                                                                             5.2                                                                              77.0                                                                              .sup. N.D..sup.b                                                                  14.9  50.9                                                                              9.5   11.7                                                                             4.3                                                                              0.001 35.5                            0650E003                                                                             7.1                                                                              88.0                                                                              N.D.                                                                              20.0  54.1                                                                              N.D.   9.1                                                                             2.2                                                                              0.001 39.5                            __________________________________________________________________________     .sup.a Kaolin was substituted for soluble ellagic acid in the assay.          .sup.b N.D. = not done.                                                  

EXAMPLE III

The products of this invention were distributed to 13 investigators foran evaluation of clinical efficacy. 33 patients in all were treated fora total of 74 bleeding episodes. All of the patients but one exhibitedvarious levels of factor VIII antibody; the patient not having factorVIII antibody is additionally reported in Example IV. The bleedingepisodes among these patients predominantly involved the joints (59.5%),while soft tissue (14.8%) and combination joint and soft tissue bleeds(5.4%) accounted for the bulk of the remainder. Of the remaining 15patients ten (13.5%) were surgically oriented, three exhibitedhematuria, one hematemesis and one intracranial bleed.

The population of total administered doses, some over multipleinfusions, ranged from 9 to 1,861 factor VIII correctional units/kg. Thedistribution of doses is further discussed below in connection withpatient prothrombin time (PT) and activated partial thromboplastin time(PTT), Tables 4 and 5. Each dosage was administered by infusion insterile water for injection. In most cases a single infusion of about 30ml was sufficient to achieve a moderate or excellent clinical response,although more than one dose was administered in a number of the bleedingepisodes.

Clinical performance of the activated PCC was subjectively evaluated byeach investigator within eight hours after the infusion. Whileevaluations were made on the overall clinical response the investigatorsfocused on hemostasis, pain relief and improvements in joint motion. Ingeneral, an "excellent" overall clinical response meant abrupt painrelief and unequivocal decrease in joint or bleed site size, usuallywithin eight hours after a single infusion. A "moderate" overallclinical response was defined as definite but slightly delayed pain andbleed site relief which, in some cases, required more than one infusion.A "fair" overall clinical response meant an unclear, but probablebeneficial effect requiring still more infusions. An overall clinicalresponse designated as "none" meant no effect relative to pain, range ofjoint motion, or degree of swelling of the bleeding site.

The overall clinical response resulting from the treatment with theproducts in the 74 bleeding episodes is summarized in Table 3. Forconvenience the factor VIII correctional activity dosage was dividedinto two groups, those above and below 50 units/kg.

                  TABLE 3                                                         ______________________________________                                        Overall Clinical Response                                                                    Patients Dosed With                                                                     greater than                                                                             less than                                                          50 U/kg    50 U/kg                                   Rating  Total   % Total  (Total)    (Total)                                   ______________________________________                                        Excellent                                                                             43      58.1     33/50 (66.0%)                                                                            10/24 (41.6%)                             Moderate                                                                              22      29.7     10/50 (20.0%)                                                                            12/24 (50.0%)                             Fair     6      8.1       5/50 (10.0%)                                                                            1/24 (4.2%)                               None     3      4.1      2/50 (4.0%)                                                                              1/24 (4.2%)                               TOTAL   74      100.0    50/74 (67.6%)                                                                            24/74 (32.4%)                             ______________________________________                                    

Of the total bleeds, 43 or 58.1% were categorized by the investigatorsas exhibiting an excellent clinical response. Combining excellentclinical responses with moderate responses resulted in favorable resultsfor 65 (87.8%) of the total bleeding episodes. Nine bleeding episodes(12.2%) were categorized by the investigators as fair or no clinicalresponse, the latter accounting for three of the nine bleeding episodes.

Table 3 further shows that of the 50 bleeding episodes receiving dosesgreater than 50 units of factor VIII correctional activity/kg, 33 or 66%exhibited an excellent clinical response while only 10 (or 41.6%) of the24 episodes dosed with less than 50 units had an excellent response.Thus it is concluded that a dosage of greater than 50 units of factorVIII correctional activity/kg is preferred for the best therapeutictreatment of bleeding episodes.

Of the three bleeding episodes where no clinical response occurred, onepatient was treated initially with relatively low doses of product (10units/kg and 22 hours later with 21 units/kg). Not until after thepatient received two additional doses of 42 units/kg (each approximately22 hours apart) in conjunction with the application of a plaster splintdid some gradual improvement take place.

The second patient exhibited slight improvement after the first of threeinfusions of 31.8 units/kg. However, the subsequent doses provded to beof no benefit and the patient was switched to factor VIII therapy. Afterthree doses of factor VIII (3,500 units) slow improvement in the bleedoccurred.

The third patient received only a small, single infusion of product(39.1 units/kg).

Since it is extremely important in the control of bleeding episodes toachieve rapid remission of symptoms the data were arranged todemonstrate the effect of single doses of the invention product. Table 4summarizes the results.

                  TABLE 4                                                         ______________________________________                                        Overall Clinical Response After A Single Dose                                          %     Patients' Initial Dose                                         Rating Total    Total  >50 U/kg   <50 U/kg                                    ______________________________________                                        Excellent                                                                            40      66.6    28    (87.5%)                                                                              12    (42.8%)                             Moderate                                                                             18      30.0    3     (9.4%) 15    (53.6%)                             Fair    1      1.7     1     (3.1%) 0     (0%)                                None    1      1.7     0     (0%)   1     (3.6%)                              TOTAL  60      100.0   32/60 (53.3%)                                                                              28/60 (46.7%)                             ______________________________________                                    

It can be seen that considerably better results are achieved with dosesof 50 U/kg or greater.

The correlation among activated PCC dose, post-infusion PTT and PTassays, and clinical response was investigated by determining theminimal and maximal PT and PTT decrease from pre-infusion topost-infusion, a span varying between 15 minutes and 2 hours. Theresults are set forth in Tables 5 and 6 as differences from controltimes, i.e., delta differences. The units are factor VIII correctionalunits.

                  TABLE 5                                                         ______________________________________                                        Change in PTT after Initial Infusion                                                 Δ PTT                                                                           Number of Clinical Responses                                   Dose Range                                                                              Range    To-    Excel-                                                                              Moder-                                        (U/kg)   (Sec.)    tal    lent  ate   Fair None                               ______________________________________                                         7.9-39.1                                                                               1.1-14.5 21     9     11    0    1                                  31.5-50.1                                                                              16.0-61.5  4     2     2     0    0                                   52.0-100.0                                                                            16.9-46.3 20     15    3     1    1                                   57.5-103.0                                                                             1.6-14.5  6     5     1     0    0                                  ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Change in PT after Initial Infusion                                                  Δ PT                                                                            Number of Clinical Responses                                   Dose Range                                                                              Range    To-    Excel-                                                                              Moder-                                        (U/Kg)   (Sec.)    tal    lent  ate    Fair                                                                              None                               ______________________________________                                         5.3-39.1                                                                              1.3-1.9    5     2     2     0    1                                   7.9-50.0                                                                              2.4-5.2   21     9     12    0    0                                   52.0-103.0                                                                            2.0-7.0   21     17    3     0    1                                   55.0-100.0                                                                            0.5-1.7    6     5     1     0    0                                  ______________________________________                                    

In conclusion, 74 bleeding episodes were treated with the product ofthis invention in 33 patients that exhibited clotting factordeficiencies, primarily factor VIII inhibitor. 71 of these 74 episodesresulted in a beneficial clinical response. 59 of the 71 required butone infusion to produce a beneficial clinical response, which occurredin 55 out of 59 cases within eight hours of the infusion. Six requiredtwo infusions and six required three or more infusions to produce abeneficial clinical response.

The greatest reduction in both the PTT and PT values occurred with dosesabove 50 units of factor VIII correctional activity/kg and were inconjunction with greater beneficial clinical results.

EXAMPLE IV

One of the patients in the clinical study reported in Example III had ahistory of factor XI deficiency. In addition, factor XI inhibitor wasobserved. This patient had a spontaneous left thigh bleed for which heinitially received one liter/day of fresh frozen plasma on hospital daysone through seven. The product of this invention was administered ondays 10 through 13. The overall clinical response after four infusionstotalling 177 factor VIII correctional units/kg was rated by theinvestigator as moderate. The investigator commented that "most likelythe patient would have lost the use of his leg due to the bleed withoutthe availability of the product."

I claim:
 1. A method for treating a patient having an inhibitor of ablood clotting factor, which comprises administering to the patient atherapeutically effective dose of an aqueous composition comprisingabout from 20 to 112 units of F-IX/ml and from 0 to about 30 units ofF-IX precursor/ml.
 2. A method for treating a patient having aninhibitor of a blood clotting factor, which comprises administering tothe patient a therapeutically effective dose of an aqueous compositioncomprising about from 1 to 50 units of F-X/ml and about from 4 to 10units of F-Xa/ml.
 3. The method of claims 1 or 2 wherein thetherapeutically effective dose is greater than about 25 factor VIIIcorrectional units/kg of patient weight.
 4. The method of claims 1 or 2wherein the aqueous composition additionally comprises greater than 1.1units of heparin/ml.
 5. The method of claims 1 or 2 wherein the aqueouscomposition additionally comprises antithrombin III.
 6. The method ofclaims 1 or 2 wherein the aqueous composition is sufficiently free offactor VIII antigen to not provoke an immune response in the patient tofactor VIII.
 7. A composition for the treatment of clotting factordeficiencies and inhibitors which comprises, in relative proportionateunits, prothrombin, about from 1-10; thrombin, less than about 0.003,factor VII, about from 37-190; factor VIIa about from 8-80; factor IX,about from 15-112; factor IX precursor, 9 to about 30; factor X, aboutfrom 1-30; and factor Xa about from 1-10.
 8. A composition for thetreatment of clotting factor inhibitors which comprises, in relativeproportionate units, prothrombin, about from 3.6-8.9; thrombin, lessthan about 0.002, factor VII, about from 37-122; factor VIIa about from25-78; factor IX, about from 20-81; factor IX precursor, 5 to about 20;factor X about from 1-25; and factor Xa about from 2-10.
 9. Acomposition for the treatment of clotting factor inhibitors whichcomprises, in relative proportionate units, prothrombin, about from3.6-5.9; thrombin, less than about 0.001, factor VII, about from 39-88;factor VIIa about from 25-60; factor IX, about from 50-80; factor IXprecursor, 5 to about 12; factor X about from 1-13; and factor Xa aboutfrom 4-10.
 10. A composition for treating clotting factor inhibitorswhich comprises about from 20-112 proportionate units of factor IX andabove 0 to about 30 units of factor IX precursor.
 11. An aqueouscomposition for treating clotting factor inhibitors which comprisesabout from 1 to 13 units of F-X/ml and about from 4 to 10 units ofF-Xa/ml.
 12. The compositions of claims 6, 7, 8 or 9 additionallycomprising greater than 1.1 proportionate units of heparin.
 13. Thecomposition of claim 12 additionally comprising antithrombin III. 14.The composition of claims 7, 8, 9 or 10 which is sufficiently free offactor VIII antigen to not provoke an immune response to factor VIII inhumans.
 15. The composition of claims 7, 8 or 9 dissolved in aqueoussolution.
 16. The composition of claim 10 dissolved in an aqueoussolution to a concentration of about from 20-112 units of factor IX/mland about 0 to about 30 units of factor IX precursor/ml.
 17. Thecomposition of claim 10 further including about from 8 to 80proportionate units of factor VIIa.
 18. The composition of claim 10containing about from 25 to 78 proportionate units of factor VIIa.